Development and remodeling of retinal blood vessels occurs through the complex and still poorly understood processes of vasculogenesis and angiogenesis. These processes are recapitulated, though imperfectly, in the context of ischemic injury associated with numerous ocular diseases, (e.g., retinopathy of prematurity, age-related macular degeneration, diabetic retinopathy, etc). The pathogenesis of these retinal neovascular disorders is driven by a pathological angiogenesis leading to the formation of abnormal vessels, which break into the vitreous, and an insufficient/limited vasculogenesis in which the cellular components of the new vessel complex originate in part, from bone-marrow (BM) derived circulating vascular progenitor cells. Components of the vascular extracellular matrix (ECM) coordinate the execution of multiple angiogenic and vasculogenic activities. Recent work in the PI's laboratory have revealed that the cysteine-rich protein 61 (Cyr61), a novel ECM-associated, heparin- and integrin-binding protein, stimulates the angiogenic phenotype of the BM-derived hematopoeitic progenitors, CD34+ cells. However, when exposed to cultured retinal pericytes, which sheath and regulate the growth of retinal capillaries, Cyr61 induces their death by anoikis suggesting a role of Cyr61 in pathological angiogenesis. The current proposal consists of a strategy to tease out these disparate activities of the Cyr61 protein both in vitro and in vivo. We will aim to determine that the multiple activities of Cyr61 are linked to its multi-modular organization consisting of 4 distinct domains: an insulin-like growth factor binding protein (IGFBPs) domain, the von Willebrand factor type C (vWFC) repeat, the thrombospondin type I (TSP1) repeat and a C-terminal (CT) cystine-knot motif. We predict that the N-terminal region containing IGFBP and/or vWFC domains with integrin-binding capabilities retains the angiogenic potential of Cyr61 vis-`-vis the CD34+ cells and promote their adhesion, migration, differentiation, and angiogenic activity. Conversely, the C-terminal region containing the TSP1 and/or CT domains induces apoptosis of retinal pericytes by virtue of its anti-adhesive properties. We will reveal these activities, in Specific Aim 1 using undifferentiated hematopoeitic CD34+ cells and cultured retinal pericytes exposed to recombinantly produced domains of Cyr61 or lentiviral vectors expressing the N-terminal or C- terminal regions. In Specific Aim 2, we will utilize the neonate mouse model of oxygen-induced retinopathy characterized by vaso-obliteration of the central retinal capillary bed to determine whether administration of either Cyr61 or Cyr61 modules promotes vascular repair by enhancing recruitment of EPCs to retinal sites of ischemic injury. Similarly, we will establish whether, BM-derived CD34+ cells, when primed with the N- terminal domains of Cyr61, integrate into damaged retinal vessels and improve vascular recovery. Our studies will help establish treatment alternatives for the diminished/limited vasculogenic capability in ischemic retinopathy and the specific contribution of truncated ECM proteins to EPC mobilization and recruitment. PUBLIC HEALTH RELEVANCE: Neovascularization of the retina is a highly prevalent and potentially blinding disorder characteristic of a variety of ocular diseases including diabetic retinopathy, age-related macular degeneration, central retinal vein occlusion, and retinopathy of permaturity. Our studies address the role of a specific component of the extracellular environment in retinal vessels and its ability to either facilitate or antagonize retinal vascular repair/rescue. Results of our studies will determine how specific portions of such extracellular compound can be used/modified to allow the formation of normally functioning retinal blood vessels and improve the pharmacotherapy of several retinal diseases.